RESUMO
Laccases are enzymes catalyzing the oxidation of a wide range of organic and inorganic substrates accompanied by molecular oxygen reduction to water. Recently, oxygen reduction by laccases has been studied by single-crystal serial X-ray crystallography with increasing absorption doses at subatomic resolution. There were two determined structures corresponding to the reduced and oxidized stable states of the laccase active site. However, the protonation of the oxygen ligands involved cannot be determined even at subatomic resolution. In the present work, the protonation of oxygen ligands in the active site of laccase for the two stable states determined in the X-ray study was explored using quantum mechanical and continuum-electrostatics calculations. This is important for understanding the reaction of the oxygen reduction mechanism in laccases. The high precision of X-ray data at subatomic resolutions allowed us to optimize the quantum mechanical calculations.
Assuntos
Lacase , Oxigênio , Lacase/metabolismo , Domínio Catalítico , Oxigênio/química , Conformação Proteica , Oxirredução , Cristalografia por Raios XRESUMO
Nucleoside phosphorylases are involved in the salvage pathways of nucleoside biosynthesis and catalyze the reversible reaction of a nucleobase with α-d-ribose-1-phosphate to yield a corresponding nucleoside and an inorganic phosphate. The equilibrium of these reactions is shifted towards nucleosides, especially in the case of purines. Purine nucleoside phosphorylase (PNP, EC 2.4.2.1) is widely used in labs and industry for the synthesis of nucleosides of practical importance. Bacterial PNPs have relatively broad substrate specificity utilizing a wide range of purines with different substituents to form the corresponding nucleosides. To shift the reaction in the opposite direction we have used arsenolysis instead of phosphorolysis. This reaction is irreversible due to the hydrolysis of the resulting α-d-ribose-1-arsenate. As a result, heterocyclic bases are formed in quantitative yields and can be easily isolated. We have developed a novel method for the preparation of cytokinins based on the enzymatic cleavage of the N-glycosidic bond of N6-substituted adenosines in the presence of PNP and Na2HAsO4. According to the HPLC analysis the conversion proceeds in quantitative yields. In the proposed strategy the ribose residue acts as a protective group. No contamination of the final products with AsO43- has been detected via HPLC-HRMS; simple analytical arsenate detection via ESI-MS has been proposed.
Assuntos
Citocininas/síntese química , Nucleosídeos/química , Adenosina/análogos & derivados , Adenosina/metabolismo , Arseniatos/química , Proteínas de Bactérias , Cromatografia Líquida de Alta Pressão , Purina-Núcleosídeo Fosforilase/metabolismo , Ribose/químicaRESUMO
We present the functional and structural characterization of the first archaeal thermostable NADP-dependent aldehyde dehydrogenase AlDHPyr1147. In vitro, AlDHPyr1147 catalyzes the irreversible oxidation of short aliphatic aldehydes at 60-85°Ð¡, and the affinity of AlDHPyr1147 to the NADP+ at 60°Ð¡ is comparable to that for mesophilic analogues at 25°Ð¡. We determined the structures of the apo form of AlDHPyr1147 (3.04 Å resolution), three binary complexes with the coenzyme (1.90, 2.06, and 2.19 Å), and the ternary complex with the coenzyme and isobutyraldehyde as a substrate (2.66 Å). The nicotinamide moiety of the coenzyme is disordered in two binary complexes, while it is ordered in the ternary complex, as well as in the binary complex obtained after additional soaking with the substrate. AlDHPyr1147 structures demonstrate the strengthening of the dimeric contact (as compared with the analogues) and the concerted conformational flexibility of catalytic Cys287 and Glu253, as well as Leu254 and the nicotinamide moiety of the coenzyme. A comparison of the active sites of AlDHPyr1147 and dehydrogenases characterized earlier suggests that proton relay systems, which were previously proposed for dehydrogenases of this family, are blocked in AlDHPyr1147, and the proton release in the latter can occur through the substrate channel.
Assuntos
Aldeído Desidrogenase/química , Aldeído Desidrogenase/metabolismo , NADP/metabolismo , Pyrobaculum/enzimologia , Domínio Catalítico , Cristalografia por Raios X , Estabilidade Enzimática , Temperatura Alta , Cinética , Modelos Moleculares , Conformação Proteica , Especificidade por SubstratoRESUMO
Laccases are members of a large family of multicopper oxidases that catalyze the oxidation of a wide range of organic and inorganic substrates accompanied by the reduction of dioxygen to water. A new laccase was isolated from the basidiomycete Coriolopsis caperata strain 0677 and its amino-acid sequence was determined. According to its physicochemical properties and spectroscopic features, the laccase from C. caperata is a high redox-potential blue laccase. Attempts to crystallize the native enzyme were unsuccessful. The copper type 2-depleted (T2D) laccase was prepared and crystallized. The structure of T2D laccase from C. caperata was solved at 1.6â Å resolution, and attempts to reconstruct the T2 copper centre were performed using Cu(+) and Cu(2+) ions. The structure of T2D+Cu(+) laccase was solved at 1.89â Å resolution. It was shown that the T2D+Cu(+) laccase structure contained four copper ions in the active site. Reconstruction could not be achieved when the T2D laccase crystals were treated with CuSO4.
Assuntos
Cobre/química , Coriolaceae/enzimologia , Lacase/química , Domínio Catalítico , Cobre/metabolismo , Coriolaceae/química , Cristalografia por Raios X , Lacase/metabolismo , Modelos Moleculares , Oxirredução , Conformação ProteicaRESUMO
Uridine phosphorylase (UP; EC 2.4.2.3), a key enzyme in the pyrimidine-salvage pathway, catalyzes the reversible phosphorolysis of uridine to uracil and ribose 1-phosphate. Expression of UP from Shewanella oneidensis MR-1 (SoUP) was performed in Escherichia coli. The high-resolution X-ray structure of SoUP was solved in the free form and in complex with uridine. A crystal of SoUP in the free form was grown under microgravity and diffracted to ultrahigh resolution. Both forms of SoUP contained sulfate instead of phosphate in the active site owing to the presence of ammonium sulfate in the crystallization solution. The latter can be considered as a good mimic of phosphate. In the complex, uridine adopts a high-syn conformation with a nearly planar ribose ring and is present only in one subunit of the hexamer. A comparison of the structures of SoUP in the free form and in complex with the natural substrate uridine showed that the subunits of the hexamer are not identical, with the active sites having either an open or a closed conformation. In the monomers with the closed conformation, the active sites in which uridine is absent contain a glycerol molecule mimicking the ribose moiety of uridine.
Assuntos
Shewanella/enzimologia , Uridina Fosforilase/química , Uridina/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Infecções por Bactérias Gram-Negativas/microbiologia , Humanos , Conformação Proteica , Shewanella/química , Shewanella/metabolismo , Uridina/química , Uridina Fosforilase/metabolismoRESUMO
Octahaem cytochrome c nitrite reductase from Thioalkalivibrio nitratireducens (TvNiR), like the previously characterized pentahaem nitrite reductases (NrfAs), catalyzes the six-electron reductions of nitrite to ammonia and of sulfite to sulfide. The active site of both TvNiR and NrfAs is formed by the lysine-coordinated haem and His, Tyr and Arg residues. The distinguishing structural feature of TvNiR is the presence of a covalent bond between the CE2 atom of the catalytic Tyr303 and the S atom of Cys305, which might be responsible for the higher nitrite reductase activity of TvNiR compared with NrfAs. In the present study, a new modified form of the enzyme (TvNiRb) that contains an additional covalent bond between Tyr303â CE1 and Gln360â CG is reported. Structures of TvNiRb in complexes with phosphate (1.45â Å resolution) and sulfite (1.8â Å resolution), the structure of TvNiR in a complex with nitrite (1.83â Å resolution) and several additional structures were determined. The formation of the second covalent bond by Tyr303 leads to a decrease in both the nitrite and sulfite reductase activities of the enzyme. Tyr303 is located at the exit from the putative proton-transport channel to the active site, which is absent in NrfAs. This is an additional argument in favour of the involvement of Tyr303 as a proton donor in catalysis. The changes in the activity of cytochrome c nitrite reductases owing to the formation of Tyr-Cys and Tyr-Gln bonds may be associated with changes in the pK(a) value of the catalytic tyrosine.
Assuntos
Citocromos a1/química , Citocromos a1/metabolismo , Citocromos c1/química , Citocromos c1/metabolismo , Ectothiorhodospiraceae/enzimologia , Nitrato Redutases/química , Nitrato Redutases/metabolismo , Tirosina/química , Domínio Catalítico , Cristalografia por Raios X , Ectothiorhodospiraceae/química , Modelos Moleculares , Nitritos/metabolismo , Fosfatos/metabolismo , Ligação Proteica , Sulfitos/metabolismo , Tirosina/metabolismoRESUMO
Uridine phosphorylase (UDP, EC 2.4.2.3), a key enzyme in the pyrimidine salvage pathway, catalyses the reversible phosphorolysis of uridine to uracil and ribose 1-phosphate. The gene expression of UDP from Shewanella oneidensis MR-1 was performed in the recipient strain Escherichia coli. The UDP protein was crystallized on earth (in the free form and in complex with uridine as the substrate) by the hanging-drop vapour-diffusion method at 296â K and under microgravity conditions (in the free form) aboard the Russian Segment of the International Space Station by the capillary counter-diffusion method. The data sets were collected to a resolution of 1.9â Å from crystals of the free form grown on earth, 1.6â Å from crystals of the complex with uridine and 0.95â Å from crystals of the free form grown under microgravity. All crystals belong to the space group P2(1) and have similar unit-cell parameters. The crystal of uridine phosphorylase grown under microgravity diffracted to ultra-high resolution and gave high-quality X-ray diffraction data.
Assuntos
Proteínas de Bactérias/química , Shewanella/enzimologia , Uridina Fosforilase/química , Cristalização , Cristalografia por Raios X , Uridina/química , Ausência de PesoRESUMO
Protealysin (PLN) belongs to the M4 family of peptidases that are commonly known as thermolysin-like proteases (TLPs). All TLPs are synthesized as precursors containing N-terminal propeptides. According to the primary structure of the N-terminal propeptides, the family is divided into two distinct groups. Representatives of the first group including thermolysin and all TLPs with known three-dimensional structures have long prosequences ( approximately 200 amino acids). Enzymes of the second group, whose prototype is protealysin, have short ( approximately 50 amino acids) propeptides. Here, we present the 1.8 A crystal structure of PLN precursor (proPLN), which is the first three-dimensional structure of a TLP precursor. Whereas the structure of the catalytic domain of proPLN is similar overall to previously reported structures of mature TLPs, it has specific features, including the absence of calcium-binding sites, and different structures of the N-terminal region and substrate-binding site. PLN propeptide forms a separate domain in the precursor and likely acts as an inhibitor that blocks the substrate-binding site and fixes the "open" conformation of the active site, which is unfavorable for catalysis. Furthermore the conserved PPL motif identified in our previous studies directly interacts with the S' subsites of the active center being a critical element of the propeptide-catalytic domain interface. Comparison of the primary structures of TLPs with short propeptides suggests that the specific features revealed in the proPLN crystal structure are typical for all protealysin-like enzymes. Thus, such proteins can be considered as a separate subfamily of TLPs.
Assuntos
Precursores Enzimáticos/química , Precursores Enzimáticos/metabolismo , Metaloendopeptidases/química , Metaloendopeptidases/metabolismo , Sequência de Aminoácidos , Domínio Catalítico , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência MolecularRESUMO
The structures of complexes of octahaem cytochrome c nitrite reductase from the bacterium Thioalkalivibrio nitratireducens (TvNiR) with the substrate sulfite (1.4 Å resolution; R(cryst) = 0.126) and the inhibitor cyanide (1.55 Å resolution; R(cryst) = 0.148) have been established. The complex with sulfite was prepared by the reduction of the protein crystal with sodium dithionite. The sulfite ion is bound to the iron ion of the catalytic haem through the S atom. The Fe-S distance is 2.24 Å. The structure of the cyanide complex with full occupancy of the ligand site was established for the first time for cytochrome c nitrite reductases. The cyanide ion is bound to the catalytic haem iron through the C atom. The Fe-C distance is 1.91 Å and the Fe-C-N angle is 171°. The sulfite reductase activity of TvNiR was measured at different pH values. The activity is 0.02â µmol of HS(-) per minute per milligram at pH 7.0; it decreases with increasing pH and is absent at pH 9.0.
Assuntos
Grupo dos Citocromos c/metabolismo , Ectothiorhodospira/enzimologia , Complexos Multiproteicos/metabolismo , Nitrito Redutases/metabolismo , Cristalografia por Raios X , Cianetos/metabolismo , Grupo dos Citocromos c/química , Inibidores Enzimáticos , Concentração de Íons de Hidrogênio , Complexos Multiproteicos/química , Nitrito Redutases/química , Ligação Proteica , Conformação Proteica , Especificidade por Substrato , Sulfitos/metabolismoRESUMO
Nucleoside phosphorylases catalyze the reversible phosphorolysis of nucleosides to heterocyclic bases, giving α-d-ribose-1-phosphate or α-d-2-deoxyribose-1-phosphate. These enzymes are involved in salvage pathways of nucleoside biosynthesis. The level of these enzymes is often elevated in tumors, which can be used as a marker for cancer diagnosis. This review presents the analysis of conformations of nucleosides and their analogues in complexes with nucleoside phosphorylases of the first (NP-1) family, which includes hexameric and trimeric purine nucleoside phosphorylases (EC 2.4.2.1), hexameric and trimeric 5'-deoxy-5'-methylthioadenosine phosphorylases (EC 2.4.2.28), and uridine phosphorylases (EC 2.4.2.3). Nucleosides adopt similar conformations in complexes, with these conformations being significantly different from those of free nucleosides. In complexes, pentofuranose rings of all nucleosides are at the W region of the pseudorotation cycle that corresponds to the energy barrier to the NâS interconversion. In most of the complexes, the orientation of the bases with respect to the ribose is in the high-syn region in the immediate vicinity of the barrier to syn â anti transitions. Such conformations of nucleosides in complexes are unfavorable when compared to free nucleosides and they are stabilized by interactions with the enzyme. The sulfate (or phosphate) ion in the active site of the complexes influences the conformation of the furanose ring. The binding of nucleosides in strained conformations is a characteristic feature of the enzyme-substrate complex formation for this enzyme group.
Assuntos
Domínio Catalítico , Nucleosídeos/química , Pentosiltransferases/química , Animais , Humanos , Pentosiltransferases/metabolismoRESUMO
This paper describes the structural analysis of the native form of laccase from Trametes hirsuta at 1.8 A resolution. This structure provides a basis for the elucidation of the mechanism of catalytic action of these ubiquitous proteins. The 1.8 A resolution native structure provided a good level of structural detail compared with many previously reported laccase structures. A brief comparison with the active sites of other laccases is given.
Assuntos
Cristalografia por Raios X , Lacase/química , Trametes/enzimologia , Domínio Catalítico , Cobre/metabolismo , Cristalização , Lacase/metabolismo , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
Laccases are enzymes that catalyze the oxidation of a wide range of organic and inorganic substrates accompanied by the reduction of molecular oxygen to water. Here, a subatomic resolution X-ray crystallographic study of the mechanism of inhibition of the laccase from the basidiomycete fungus Steccherinum murashkinskyi by chloride and fluoride ions is presented. Three series of X-ray diffraction data sets were collected with increasing doses of absorbed X-ray radiation from a native S. murashkinskyi laccase crystal and from crystals of complexes of the laccase with chloride and fluoride ions. The data for the native laccase crystal confirmed the previously deduced enzymatic mechanism of molecular oxygen reduction. The structures of the complexes allowed the localization of chloride and fluoride ions in the channel near the T2 copper ion. These ions replace the oxygen ligand of the T2 copper ion in this channel and can play the role of this ligand in the enzymatic reaction. As follows from analysis of the structures from the increasing dose series, the inhibition of laccases by chloride and fluoride anions can be explained by the fact that the binding of these negatively charged ions at the position of the oxygen ligand of the T2 copper ion impedes the reduction of the T2 copper ion.
Assuntos
Cloretos/metabolismo , Cobre/metabolismo , Fluoretos/metabolismo , Lacase/química , Basidiomycota/enzimologia , Domínio Catalítico , Cristalografia por Raios X/métodos , Ligantes , Modelos Moleculares , Oxirredução , Oxigênio/metabolismo , Conformação Proteica , Imagem Individual de Molécula/métodosRESUMO
Laccases are multicopper oxidases that catalyze oxidation of a wide range of organic and inorganic substrates accompanied by the reduction of dioxygen to water. The physicochemical and catalytic properties of two new fungal laccases from basidiomycetes Antrodiella faginea (AfL) and Steccherinum murashkinskyi (SmL) with middle redox potential of the T1 copper site were studied. The X-ray structures of AfL and SmL were solved at 1.75â¯Å and 0.95â¯Å, respectively. The oxidized state of copper ions in the active site was observed in AfL structure, while the mixture of oxidized and reduced states was observed in SmL structure. These oxidized and reduced states relate to the position of copper ions, their coordination, and nature and position of oxygen ligands. Comparative analysis of the T1 site environment of laccases with known structure allowed us to highlight the six types of the secondary coordination sphere of the T1 copper. The solvent accessible surface area of the conservative region of the secondary coordination sphere of the T1 copper correlates with its the redox potential. It was shown that the laccase classification by the structure of the T1 copper secondary coordination sphere is in agreement to ecophysiological behavior of laccase producing fungi.
Assuntos
Basidiomycota/enzimologia , Lacase/química , Conformação Proteica , Catálise , Domínio Catalítico , Cobre/química , Cristalografia por Raios X , Ligantes , Modelos Moleculares , Oxirredução , Oxigênio/química , Relação Estrutura-Atividade , Água/químicaRESUMO
A highly active cytochrome c nitrite reductase from the haloalkaliphilic sulfur-oxidizing non-ammonifying bacterium Tv. nitratireducens strain ALEN 2 (TvNiR) was isolated and purified to apparent electrophoretic homogeneity. The enzyme catalyzes reductive conversion of nitrite and hydroxylamine to ammonia without release of any intermediates, as well as reduction of sulfite to sulfide. TvNiR also possesses peroxidase activity. In solution TvNiR exists as a stable hexamer with molecular mass of about 360kDa. Each TvNiR subunit with molecular mass of 64kDa contains, as defined from spectral properties and sequence analysis, eight c-type haems. Seven of them are coordinated by the characteristic CXXCH motifs for haem c binding, while one is bonded by the unique CXXCK motif. So far, this motif coordinating the catalytic haem was found only in bacterial cytochrome c nitrite reductases (ccNiRs). All the residues essential for catalysis in the known ccNiRs were also identified in TvNiR. However, TvNiR is only distantly related to known bacterial ammonifying dissimilatory ccNiRs, sharing no more than 20% homology.
Assuntos
Citocromos a1/química , Citocromos a1/metabolismo , Citocromos c1/química , Citocromos c1/metabolismo , Nitrato Redutases/química , Nitrato Redutases/metabolismo , Sequência de Aminoácidos , Ectothiorhodospiraceae/enzimologia , Heme/análise , Cinética , Dados de Sequência Molecular , Estrutura Quaternária de Proteína , Alinhamento de Sequência , EspectrofotometriaRESUMO
Biochemical analysis of enantioselective short-chain alcohol dehydrogenase from the hyperthermophilic archaeon Thermococcus sibiricus (TsAdh319) revealed unique polyextremophilic properties of the enzyme - half-life of 1 h at 100 °C, tolerance to high salt (up to 4 M) and organic solvents (50% v/v) concentrations. To elucidate the molecular basis of TsAdh319 polyextremophilicity, we determined the crystal structure of the enzyme in a binary complex with 5-hydroxy-NADP at 1.68 Å resolution. TsAdh319 has a tetrameric structure both in the crystals and in solution with an intersubunit disulfide bond. The substrate-binding pocket is hydrophobic, spacious and open that is consistent with the observed promiscuity in substrate specificity of TsAdh319. The present study revealed an extraordinary number of charged residues on the surface of TsAdh319, 70% of which were involved in ion pair interactions. Further we compared the structure of TsAdh319 with the structures of other homologous short-chain dehydrogenases/reductases (SDRs) from thermophilic and mesophilic organisms. We found that TsAdh319 has the highest arginine and aspartate + glutamate contents compared to the counterparts. The frequency of occurrence of salt bridges on the surface of TsAdh319 is the highest among the SDRs under consideration. No differences in the proline, tryptophan, and phenylalanine contents are observed; the compactness of the protein core of TsAdh319, the monomer and tetramer organization do not differ from that of the counterparts. We suggest that the unique thermostability of TsAdh319 is associated with the rigidity and simultaneous "resilience" of the structure provided by a compact hydrophobic core and a large number of surface ion pairs. An extensive salt bridge network also might maintain the structural integrity of TsAdh319 in high salinity.
Assuntos
Álcool Desidrogenase/química , Proteínas Arqueais/química , Estrutura Quaternária de Proteína , Thermococcus/enzimologia , Álcool Desidrogenase/genética , Álcool Desidrogenase/metabolismo , Sequência de Aminoácidos , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Interações Hidrofóbicas e Hidrofílicas , Ferro/química , Ferro/metabolismo , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , NADP/química , NADP/metabolismo , Ligação Proteica , Dobramento de Proteína , Multimerização Proteica , Estrutura Secundária de Proteína , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Temperatura , Thermococcus/genéticaRESUMO
Bacterial pentaheme cytochrome c nitrite reductases (NrfAs) are key enzymes involved in the terminal step of dissimilatory nitrite reduction of the nitrogen cycle. Their structure and functions are well studied. Recently, a novel octaheme cytochrome c nitrite reductase (TvNiR) has been isolated from the haloalkaliphilic bacterium Thioalkalivibrio nitratireducens. Here we present high-resolution crystal structures of the apoenzyme and its complexes with the substrate (nitrite) and the inhibitor (azide). Both in the crystalline state and in solution, TvNiR exists as a stable hexamer containing 48 hemes-the largest number of hemes accommodated within one protein molecule known to date. The subunit of TvNiR consists of two domains. The N-terminal domain has a unique fold and contains three hemes. The catalytic C-terminal domain hosts the remaining five hemes, their arrangement, including the catalytic heme, being identical to that found in NrfAs. The complete set of eight hemes forms a spatial pattern characteristic of other multiheme proteins, including structurally characterized octaheme cytochromes. The catalytic machinery of TvNiR resembles that of NrfAs. It comprises the lysine residue at the proximal position of the catalytic heme, the catalytic triad of tyrosine, histidine, and arginine at the distal side, channels for the substrate and product transport with a characteristic gradient of electrostatic potential, and, finally, two conserved Ca(2+)-binding sites. However, TvNiR has a number of special structural features, including a covalent bond between the catalytic tyrosine and the adjacent cysteine and the unusual topography of the product channels that open into the void interior space of the protein hexamer. The role of these characteristic structural features in the catalysis by this enzyme is discussed.
Assuntos
Proteínas de Bactérias/química , Citocromos a1/química , Citocromos c1/química , Ectothiorhodospiraceae/enzimologia , Nitrato Redutases/química , Estrutura Quaternária de Proteína , Sequência de Aminoácidos , Azidas/metabolismo , Cristalografia por Raios X , Heme/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Nitritos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Alinhamento de SequênciaRESUMO
Substrate specificity of E. coli thymidine phosphorylase to pyrimidine nucleoside modified at 5'-, 3'-, and 2'-positions of sugar moiety has been studied. Equilibrium (K(eq)) and kinetics constants of phosphorolysis reaction of nucleosides were measured. The most important hydrogen bonds in enzyme-substrate complex have been determined.